Immunotherapy of tumor with monoclonal antibody against the 17-1A antigen

ABSTRACT

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

BACKGROUND

[0001] The tumoricidal activity of the murine monoclonal antibody 17-1Ahas been characterized in the nude mouse and in humans. See, e.g.,Herlyn, D. and Koprowski, H. (1982) “IgG2a Monoclonal Antibodies InhibitHuman Tumor Growth Through Interaction with Effector Cells” Proc. NatlAcad. Sci. USA 79, 4761-4765. Several cases have been reported where theadministration of Mab 17-1A resulted in a partial or complete regressionof metastatic colorectal or pancreatic carinomas. See Sears, H. F. etal. (1984) “Effects of monoclonal antibody immunotherapy on patientswith gastrointestinal adonocarcinoma. ” J. Biol. Resp. Mod. 3, 138-150;Sears, H. F., et al., “Phase II Clinical Trial of a Murine MonoclonalAntibody Cytotixic for Gastrointestinal Adenocarcinoma” (1985) CancerRes. 45: 5910-5913. Generally, the antibody has been administered assingle administration of 500 μg or less.

SUMMARY OF THE INVENTION

[0002] This invention pertains to a method of immunotherapy ofgastrointestinal tumors employing multiple, high doses of murinemonoclonal antibody against the gastrointestinal tumor-associatedantigen 17-1A. The method comprises administering to a patient afflictedwith gastrointestinal tumor, murine monoclonal antibody against theantigen 17-1A in multiple, sequential doses of about 100 mg or more fora total overall dose of from about 0.1 to about 5 grams. Each dose ofthe murine antibody can be administered at one to three day intervals upto weekly intervals to achieve and maintain a “continuous” high level ofcirculating antibody. Mixtures (“cocktails”) of two or more murineanti-17-1A antibodies can be given. The multiple, high dose therapy canbe performed as adjuvant therapy to chemotherapy, radiotherapy orsurgery.

[0003] The high dose murine antibody therapy is well tolerated inpatients. Further, the anti-murine antibody response which generallydevelops in treated humans, surprisingly does not alter significantlythe plasma half life of the murine antibody on repeat administration.Thus, high blood levels of antibody can be achieved with sequentialinjections of high doses to enhance transit of the antibody fromintravascular space into the tumor bed and thus provide higherconcentrations of the therapeutic antibody to the locus of action.

DETAILED DESCRIPTION OF THE INVENTION

[0004] This invention pertains to therapy of gastrointestinal tumorswith repeated, high dose of murine antibody against the 17-1A antigenassociated with most gastrointestinal tumors. This therapeutic approachis based upon several findings. Murine anti-17-1A antibody administeredin multiple, high dose is generally well-tolerated by patients. The mostcommon side effect is mild gastrointestinal symptoms. Allergicresponses, however, do limit repeat therapy in some patients. Inaddition, although a human anti-murine antibody response is generallyevoked by the murine antibody, the response does not drastically effectthe pharmacokinetics of the administered murine antibody. This indicatesthat sequential, high doses of antibody can be given to achieve andmaintain a continuous high plasma level of antibody. Maintaining a highcirculating level of antibody optimizes transit of the antibody fromintravascular space into the tumor, thereby enhancing access of theantibody to the tumor for more effective anti-tumor action. Further,sustained, high blood levels lead to a prolonged, higher concentrationof antibody at the locus of action for more effective antibody dependentcell mediated cytolysis of the tumor cells.

[0005] According to the method of this invention, murine antibodyagainst the 17-1A antigen is administered to patients afflicted withgastointestinal tumors in multiple doses of about 100 mg or more,preferably about 400 mg-1 gram, for a total dose of about 0.1 to 5grams, preferably 1 to 5 grams. The antibody is administeredparenterally preferably by intraveneous infusion. The antibody isgenerally administered suspended in a physiologically acceptable vehiclee.g. normal saline. The antibody doses can be given over intervals of1-3 days to intervals of about a week. The dose regimen for anindividual patient will depend, inter alia, on the patient's clinicalstatus and on his ability to tolerate the dose without detrimentalallergic or anaphylactic reaction. The objective is to provide antibodyat fractional doses which yields a sustained, high plasma level ofantibody over the course of therapy to provide increased access of theantibody to the tumor site.

[0006] Murine antibodies against 17-1A can be administered individuallyor in mixtures (cocktails) of two more murine anit-17-1A antibodies.Preferably, anti-17-1A antibody having different epitopic specificityfor 17-1A is employed in the combination in order to increase anti-tumoractivity in an additive or synergistic fashion. Murine antibodies can beselected from the original 17-1A antibody or other murine antibodieswhich recognize similar or different epitopes of the 17-1A antigen, suchas the M72, M74, M77 and M79 antibodies described below.

[0007] Murine antibody against 17-1A antigen can be used in passiveimmunotherapy of tumors of the gastrointestinal tract with which the17-1A antigen is associated. Examples are gastrointestinaladenocarcinoma, colorectal carcinoma and pancreatic carcinoma. Themurine antibody treatment can be adjuvant to other forms of therapy,including chemotherapy, radiotherapy and/or surgery. In particular,murine antibody therapy can be useful as adjuvant therapy directedagainst micro- or mini-metastases which are not amenable to surgicalremoval.

[0008] The invention is further illustrated by the followingexemplification.

[0009] Exemplification

[0010] A trial was conducted in 20 patients to examine patient toleranceto repeated high doses in 17-1A, examine its pharmacokinetics onrepeated administration and to characterize the human immune response(antibody) to this mouse immunoglobulin.

[0011] Patient Population

[0012] Twenty patients with gastrointestinal malignancy (17 colon; 2gastric; and 1 pancreatic) were selected on the basis that they hadmetastatic disease with small-moderate tumor burden; performance statusgreater than 70% (Karnofsky scale) and objectively measurable disease.Studies were not done to document 17-1A reactivity with individualpatient tumor specimens. Seven out of 20 patients had received priorchemotherapy while 13/20 had no prior therapy for metastatic disease.

[0013] Treatment Protocol

[0014] The study was conducted the Clinical Research Unit, ComprehensiveCancer Center, University of Alabama at Burmingham. All antibodyinfusions utilized a total dose of 400 mg of 17-1A diluted in 250 ml ofnormal saline infused over 30 minutes with careful monitoring of vitalsigns. All infusions were preceded by an intravenous test dose of 0.7 mgfollowed by 30 minutes of monitoring prior to administration of the fulldose infusion. The protocol involved the accrual of 4 groups of 5patients each who would receive progressively increasing numbers ofweekly infusions, i.e. Group 1-5 patients—single infusion; Group 2-5patients with 2 infusions—Day 1 and 8; Group 3-5 patients with 3infusions—Day 1, 8 & 15; Group 4-5 patients with 4 infusions—Day 1, 8,15 & 22. Because of toxicity noted in Group 3 patients, no patientsreceived 4 infusions, but these 5 patients were added to Group 2 and had2 infusions of therapy (Day 1 & 8). All patients were followed for 6weeks, following their last infusion with weekly monitoring ofurinalysis, liver and renal function, blood counts and clinicalevaluation.

[0015] Pharmacokinetics

[0016] Pharmacokinetic analysis was done on the first 5 patients at thetime of their single infusion (no prior exposure to 17-1A), the tenpatients who had two infusions were studied at the time of their secondinfusion (one prior exposure to 17-1A) and the group 3 patients werestudied at the time of their third infusion (two prior exposures to17-1A). For pharmacokinetics, blood samples were drawn prior toinfusion, immediately at conclusion of infusion and at ½, 1, 2, 4, 12,24, 48, 72 hours and 86 hours. Spot samples were drawn at pre-therapy,1, 24 & 48 hours post-therapy on infusions not undergoing a fullpharmacokinetic study to confirm the general pattern of mouseimmunoglobulin disappearance. The plasma level of 17-1A was quantitatedusing a solid phase radiometric sandwich assay utilizing latex beadscoated with rabbit anti-mouse gammaglobulin and radiolabeled (¹²⁵I)affinity purified goat antimouse IgG, F(ab′)₂. The concentration of17-1A in plasma was quantitated by the amount of latex particle bindingof radiolabeled anti-mouse IgG, F(ab′)₂ as compared to a standard curveof known concentrations of 17-1A diluted in normal plasma. Thesensitivity of this assay was 1.0 ng/ml.

[0017] Human Anti-mouse Antibody (HAMA) Response

[0018] Serum samples were drawn on each patient prior to each infusionand then weekly ×6. The assay used to determine the presence of humananti-17-1A was a “double antigen” system (Addison, G. and Hale, C.,Horm. Metab. Res. 3, 59-60 (1971)) using the concurrent incubation of17-1A coated latex beads, 100 ul of test plasma of 1 ug of radiolabeled(¹²⁵I) 17-1A (specific activity of 300-400 cpm/ng). The samples wereincubated 90 minutes at room temperature and the radioactivityassociated with the beads determined by centrifugation of the beadsthrough Percoll as previously described (Lobuglio, A. et al., New Engl.J. Med. 309, 459-463 (1983)). The cpm of ¹²⁵I-17-1A bound to the beadsby plasma was converted to ng of 17-1A/ml of plasma by using the knownspecific activity of ¹²⁵I-17-1A. This assay obviously detects anymolecule with more than one binding site for 17-1A (IgG and IgM). Assayresults in normal individuals and cancer patients prior to 17-1Aexposure were 5±4 ng/ml plasma (n=54) with values ranging from 0 to 16ng/ml. Values greater than 20 ng/ml were classified as an antibodyresponse.

[0019] 17-1A Monoclonal Antibody

[0020] The monoclonal antibody was provided by Centocor, Inc. as apurified suspension of 10 mg/ml in normal saline. It was stored at 4° C.prior to use. The protocol was carried out under Centocor sponsored IND(#2168).

[0021] Toxicity

[0022] The adverse effects of 17-1A administration are summarized inTable 1. TABLE 1 TOXICITY ASSOCIATED WITH 17-1A INFUSION I. Single dose(400 mg) - 5 patients 3/5 - none 2/5 - G.I. symptoms II. Two weeklydoses (400 mg) - 10 patients 4/10 - none 5/10 - G.I. symptoms 1/10 -flushing/tachycardia III. Three weekly doses (400 mg) - 5 patients 3/5 -none 2/5 - G.I. symptoms and anaphylaxis (third dose)

[0023] Ten of 20 patients had no adverse effects including 4 patientswho received two infusions and 3 patients who received three infusions.The most frequently observed side effect was gastrointestinal (9/20patients) with nausea and vomiting (4 patients) or diarrhea with orwithout cramps (7 patients). The symptoms usually began within an hourof infusion and lasted less than 24 hours. They were of modest-moderateseverity and readily controlled with anti-emetics of anti-diarrheamedications. The frequency of gastrointestinal symptoms was not relatedto the number of 17-1A infusions. One patient had an episode of flushingand tachycardia in the midst of her second infusion which disappeared bysimply slowing the infusion rate. This patient had no other adverseeffects with the infusion nor with her prior infusion.

[0024] Two patients had serious adverse effects. Both patients hadnausea and vomiting associated with their first and second infusions(Day 1 & 8). They tolerated their test dose of 17-1A on Day 15 withoutadverse effects over 30 minutes of observation. The treatment infusionswere then begun and both developed dyspnea, tachycardia and hypotensionjudged to be an anaphylactic reaction. Both infusions were immediatelystopped (less than 10% of dose given) and patients responded well totherapy with corticosteroids, epinephrine and antihistamines. No patientin the study developed abnormalities of urinalysis, complete bloodcount, renal or hepatic function.

[0025] Pharmacokinetics

[0026] The serial plasma 17-1A levels on each patient were analyzed andfound to fit will with a 1 compartment model of plasma disappearance.The results for peak plasma concentration, plasma half-life and areaunder the curve are summarized in Table 2. TABLE 2 PHARMACOKINETICS17-1A (400 mg) IN MAN Peak Conc. Half-life AUC Prior Antibody (ug/ml)(hours) (hrs-ug/ml) None (n = 5) 139 ± 8 15 ± 2 3013 ± 175 One (n = 10)141 ± 5 14 ± 1 2828 ± 93  Two (n = 3) 108 ± 2 24 ± 2 3771 ± 81 

[0027] The two patients who had anaphylactic reactions did not receivetheir full third dose of 17-1A and therefore had no pharmacokineticstudy. Thus, only 3 patients made up the group with two prior exposuresto 17-1A. The results are similar for all 3 groups of patients. Thethree patients studied on their third infusion had a somewhat lowerserum peak concentration of 17-1A and a somewhat longer mean plasmahalf-life than the groups of patients with a single or second infusion.Interpretation is limited since the differences were modest and thegroup was made up of a small number of patients.

[0028] Human Anti-mouse Antibody (HAMA) Response

[0029] The patients' serum prior to therapy had little or no detectableability to bind ¹²⁵I-17-1A coated beads. As summarized in Table 3,almost all patients developed HAMA within 29 days of their first 17-1Aexposure (17/20). The majority (11/20) had HAMA by Day 8 with 8/11having values of greater than 100 ng/ml and 2/11 having values ofgreater than 1000 ng/ml. Peak values were generally noted on Day 15 or22 with values falling by day 29 and beyond. Patients who received one,two or three exposures to 17-1A had similar degrees by HAMA response assummarized in Table 4. TABLE 3 HUMAN ANTI-MOUSE ANTIBODY (HAMA)RESPONSE* Pre-therapy  0/20 had antibody (range - 0-15 ng/ml) Day 811/20 had antibody (range - 36-1106 ng/ml) Day 15 15/20 had antibody(range - 27-5598 ng/ml) Day 22 14/20 had antibody (range - 60-5046ng/ml) Day 29 15/20 had antibody (range - 23-4900 ng/ml) No antibody 3/20

[0030] TABLE 4 DEGREE OF HUMAN ANTI-MOUSE ANTIBODY (HAMA) RESPONSE*Exposures Very High (>1000) Moderate (40-999) Poor/none (<40) Single 3(60%) 1 (20%) 1 (20%) Double 4 (40%) 4 (40%) 2 (20%) Triple 2 (40%) 2(40%) 1 (20%)

[0031] The two patients with anaphylactic reactions were interesting.They has HAMA levels of 1055 and 264 ng/ml on Day 8 and 1716 and 3745ng/ml on Day 15, respectively. They tolerated their infusions ofantibody on Day 8 without adverse effect except for nausea and vomiting(similar to what they had on Day 1 infusion) but had anaphylacticreactions on Day 15 at the time of their third infusion. A total of 11infusions were administered to patients when their HAMA levels weregreater than 20 ng/ml (elevated) with five having no side effects, threegastrointestinal symptoms, one flushing/tachycardia and two anaphylacticreactions. No patients developed fever, proteinuria or renal impairment.

[0032] It was also interesting that in nine of these 11 infusions,adequate plasma samples were available to determine peak plasmaconcentration and plasma half-life of 17-1A antibody. These values werenot substantially different than infusions in the absence of detectableHAMA.

[0033] Discussion This phase I/II study of repeated administration of400 mg 17-1A monoclonal antibody provides several observations. Ingeneral, the administration of antibody was well tolerated in patientsreceiving one or two infusions. The mild gastrointestinal symptoms wereclearly related to antibody infusion and were not a serious clinicalproblem. The pathogenesis of these symptoms is not know but does notseem related to an allergic reaction since they occurred just asfrequently during a patient's first infusion as compared to thirdinfusion. They may be related to the ability of this antibody of bind tonormal gastrointestinal mucosa (Sears, H., et al., Surg. Res. 31,145-150 (1981)). Two of five patients receiving three weekly infusionsof 17-1A had anaphylactic reactions. This frequency of a potentiallylifethreatening allergic reaction precluded our testing a four-doseschedule (weekly) and would deter treatment schedules requiring antibodyadministration on Day 15.

[0034] The pharmacokinetic studies indicate that this dose of antibodycan achieve plasma concentrations of 100-200 ug/ml with a plasmadisappearance curve approximating observations with other mousemonoclonal antibodies (radiolabeled) administered at much lower doses(Pimm, M., et al., J. Nucl. Med. 26, 1011-1023 (1985) and Rosenblum, M.,et al., Cancer Res. 45, 2382-2386 (1985)). This plasma half-life resultsin plasma concentrations of less than 1 ug/ml by day 8. Thus,maintenance of a substantial plasma concentration of 17-1A would requireadministration more frequently than weekly. Prior studies (Pimm, M., etal., J. Nucl. Med. 26, 1011-1023 (1985) and Larson, S., et al., J. Nucl.Med. 24:123-129 (1983)) have suggested that the appearance of HAMAresponse is associated with a dramatic alteration in circulating levelsof mouse Ig. Our failure to observe this phenomenon is somewhatsurprising. However, it should be noted that our antibody measurementsare expressed in terms of ng 17-1A bound/ml plasma with an infusion of17-1A which readily achieves concentrations of 100-200 ug/ml in thecirculation. We are currently modifying our HAMA assay to allowquantitation of total circulating HAMA. This may clarify whether thepatient's total circulating HAMA is able to bind only a small fractionof this large circulating dose of 17-1A.

[0035] We did not find evidence of pre-existing human anti-mouseantibody (17-1A) prior to antibody infusion as reported by others(Schroff, R., et al., Cancer Res. 45, 879-885 (1985)). We initiallyattempted to assay for human anti-mouse antibody using an assay whichdetected human immunoglobulin binding to 17-1A coated beads usingradiolabeled monoclonal mouse anti-human Fc antibody. We found thatnormal individuals and cancer patients prior to monoclonal antibodyinfusion had varying quantities of human immunoglobulin which boundnon-specifically to 17-1A coated beads. This binding did not haveclassic competitive inhibition by soluble antigen (17-1A) and was judgedto be a non-specific phenomenon and not antibody. In contrast,post-immunization plasma immunoglobulin binding to 17-1A coated beadswas readily inhibited by soluble antigen (17-1A). Thus, we believe thatthe double antigen assay system used in this study more clearly reflectsimmune response to 17-1A. Despite the administration of one or morelarge doses of 17-1A, human antibody response to this protein was promptwith antibody frequently detectable by Day 8 and appreciable levels ofantibody achieved by Day 15 & 22. Further studies are underway tocharacterize this antibody response in regards to immunoglobulinsubclass and anti-idiotype.

BIOCHEMICAL AND EPITOPE ANALYSIS OF 17-1A ANTIGEN/PRODUCTION OFMONOCLONAL ANTIBODIES AGAINST 17-1A. Materials and Methods

[0036] Cells and Tissues

[0037] The human colon carcinoma cell lines DLD-1 and WiDr wereobrtained form the American Type Culture Collection, Rockville, Md. Thecolon carcinoma line HT-29 was kindly provided by Dr. J. Fogh, SloanKettering Institute for Cancer Research, NY. Human tissues weresnap-frozen in liquid nitrogen-cooled isopentane immediately aftersurgical removal.

[0038] Radiolabelling and Immunoprecipitation

[0039] Cells (5×10⁷) were surface-labelled by lactoperoxidase-mediatediodination using ¹²⁵I as described. Pink, J. R. L. and Ziegler, A.(1979) in: Research Methods in Immunology Academic Press, N.Y. pp.169-180. Immunosorbents prepared by binding MAbs to protein A-Sepharose(Sigma, St. Louis, Mo.) were added to labelled cell lysates at 4° C. for2 hr. Bound material was eluted by boiling in sample buffer and analyzedby SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according toLaemmli. Laemmli, U.K. (1970) Nature, 227: 680-685.

[0040] Selection of MAbs

[0041] Colon carcinoma tissue was obtained from a 51 year old femalepatient undergoing lobectomy of the liver because of a solitarymetastasis. The 17-1A positive tumor tissue was carefully isolated,minced, homogenized and plasma membranes were purified as described byTouster et al. (1970) J. Cell Biol. 47 604-618. Fusion of the myelomaP3×63Ag8.653 and spleen cells from (C57BL/6×Balb/c)F1 female mice wascarried out using standard procedures (Galfre et al., (1977) Nature 266:550-552) 3 days after a single i.p. injection of colon carcinoma plasmamembrane preparations corresponding to 3 mg of protein together withBordetella pertussis adjuvant. After fusion the cells were plated in HATselective medium (hypoxanthine, aminopterin, thymidine) in 96-wellmicrotiter plates containing mouse peritoneal macrophages. Supernatantsof hybrids were screened by immunoperoxidase staining of frozen tissuesections derived from the liver metastasis taken for immunisation.Antibodies reacting with the colon carcinoma cells of the metastasis andthe bile ducts in the adjacent liver, but not with hepatocytes, werefurther tested on a panel of non-malignant epithelial tissues listed inTable 5. Antibodies showing a 17-1A-like staining pattern in thiscontext were cloned at least two times by limiting dilution. TABLE 5DISTRIBUTION OF 17-1A ANTIGEN IN NORMAL EPITHELIAL TISSUES¹ Numbertested/ Tissue Number positive Remarks Colon 14/14 strong staining ofmucosa Small intestine 5/5 strong staining of mucosa Stomach 6/9occasional weak staining of restricted areas Gall bladder 1/1 strongstaining of mucosa Pancreas 1/1 strong staining of acini, ducts andislets of Langerhans Liver 6/6 strong staining of bile ducts;hepatocytes negative Kidney 2/2 Loops of Henle moderately, distaltubules strongly stained; proximal tubules, glomeruli negative Lung 2/2strong staining of bronchi; alveoli moderately stained Thyroid gland 4/4strong staining of follicular epithlium Mammary gland 3/3 strongstaining of lobules and ducts Thymus 0/1 Skin 2/2 strong staining ofsweat glands; epidermis negative

[0042] Immunoblotting

[0043] Immunoprecipitates were separated by SDS-PAGE,electrophoretically transferred to nitrocellulose membranes (Schleicher& Schull, Dassel, FRG) according to Towbin et al. (1979) Proc. Natl.Acad. Sci. USA 76: 4350-4354, and transferred antigen was visualized byan indirect immunoperoxidase technique, Holzmann, B. et al. (1985) J.Exp. Med. 161: 366-377.

[0044] Flow cytometric analysis

[0045] HT-29 cells were preincubated on ice with MAbs M72, M74, M77 orM79 as unconcentrated, 10×, or 50× concentrated supernatant, followed byincubation with biotinylated 17-1A antibody (10 ug/ml) andavidin-phycoerythrin (Becton-Dickinson, Mountain View, Calif.). Thefluorescence profile was analyzed with an EPICS-V (Coulter Electronics,Hialeah, Fla.).

[0046] Immunohistochemistry

[0047] Frozen tissue sections were prepared and stained by an indirectimmunoperoxidase technique essentially as described elsewhere.Gottlinger et al., (1985) Int. J. Cancer 35: 199-205. Briefly, air-driedsections (7 um) were fixed in acetone for 10 min., incubated with MAb(10 ug/ml or undiluted supernatant) for 30 min., washing in PBS andexposed for 30 min. to peroxidase-conjugated rabbit anti-mouse Igantiserum (Dianova, Hamburg, FRG) diluted 1/200 in PBS containing 20%human serum. After washing in PBS the sections were incubated for 20min. in 0.004% 3-amino-9-ethylcarbazole in 0.02 M barbital buffer, pH7.4, containing 0.001% H₂O₂ and subsequently counterstained with Mayer'shemalum.

RESULTS Biochemical Analysis of the 17-1A Ag

[0048] Since the original biochemical analysis of the 17-1A Ag was onlymade for one human tumor cell line (HT-29), we investigated the natureof the 17-1A Ag expressed on two additional human colon carcimona linesby surface iodination. Precipitation with MAb 17-1A revealed anidentical single protein band in the three cell lines, DLD-1, WiDr andHT-29, which migrated with an apparent molecular weight of 37 kD in anSDS-PAGE system. As judged from fluorographic intensity, the amounts ofantigen precipitable from the three cell lines were quite variable, withthe colon carcinoma line DLD-1 providing the highest amount ofradiolabelled antigen. Under reducing conditions, in which2-mercaptoethanol was added to the precipitates prior toelectrophoresis, a distinct band of 33 kD was obtained from all threecell lines. In addition, a major component of about 40 kD component wasalso found occasionally in HT-29 lysates. This band was apparentlyabsent or could not be precipitated from WiDr cells.

[0049] Incubation of HT-29 cells with tunicamycin (2 ug/ml) for 24 hrresulted in the appearance of a new band of 30 kD under non-reducing andof 26 kD under reducing conditions, indicating that the 17-1A Agcontains 2 N-linked glycosylation sites. The glycoprotein nature of the17-1A Ag was further substantiated by treatment of 17-1A precipitateswith neuraminidase, which resulted in a slight but distinct reduction ofthe apparent molecular weight.

Epitope Analysis of the 17-1A Ag by New MAbs

[0050] Four new MAbs directed against the 17-aA Ag (M72, M74, M77, M79)were obtained by screening supernatants of hybridomas generated frommice immunized with membrane preparations from colon carcinomametastases for a 17-1A-like reactivity on frozen tissue sections. Allfour antibodies precipitate proteins of identical molecular weight asseen with the original 17-1A antibody. In order to verify the identityof the antigens recognized by the new MAbs an extensive immunoblottinganalysis was performed, whereby 17-1A immunoprecipitates weretransferred to nitrocellulose paper after separation by SDS-PAGE andtested with the four new reagents. As shown previously, the four newantibodies bound to a 37 kD protein was not obtained withisotype-matched control MAbs. To analyze the epitope specificity of thefour new MAbs cross-blocking experiments were carried out. In a flowcytometry analysis the binding of a biotinylated 17-1A antibody to HT-29cells was completely blocked by preincubation of the tumor cells withMAbs M72 and M74 (Table 6). In contrast, MAbs M77 and M79 showed nosignificant blocking activity for the biotinylated 17-1A antibody at allconcentrations tested. We further analyzed whether the epitopespecificty could be related to the idiotypes of the antibodies. All MAbswere analyzed for reactivity with an anti-idiotypic antiserum generatedagainst the 17-1A antibody in goats (kindly provided by Dr. DorothyHerlyn, The Wistar Institute, Philadelphia). The anti-idotypic antiserumstrongly reacted with the two cross-blocking MAbs (M72 and M74), but wascompletely unreactive with the MAbs M77 and M79 (Table 6). These datasuggest that mAbs 17-1A, M72 and M74 recognize the same or closelyrelated epitopes on the 37 kD glycoprotein, while MAbs M77 and M79define additional epitopes on this antigen. TABLE 6 IDIOTYPE EXPRESSIONAND CROSS-BLOCKING ACTIVITY OF DIFFERENT MABS DIRECTED AGAINST THE 17-1AANTIGEN 17-1A M72 M74 M77 M79 Reactivity with +^(a) ++ + − − goatanti-Id 17-1A Cross-blocking +^(b) ++ ++ − − activity

Tissue Distribution of the 17-1A Ag

[0051] Using an indirect immunoperoxidase technique the 17-1A Ag wasidentified in various normal human organs and in a variety of humancarcinomas. Concomitantly the four new MAbs were analyzed on paralleltissue sections. With respect to expression of the 17-1A Ag on colontissue, it was found that normal mucosa was stained to a similar degreeas colon carcinoma tissue in the fourteen patients examined. The 17-1AAg was clearly detectable on the epithelial lining of the smallintestine, the gall bladder, the bronchi and a variety of glandularstructures, including the tyroid, the mammary gland, sweat glands andthe exocrine as well as endocraine pancreas. Furthermore, the 17-1A Agwas found to be expressed in the kidney on distal tubules and the loopsof Henle and in the liver by bile ducts, but not by hepatocytes.

[0052] In the stomach, normal mucosa usually showed a faint stainingrestricted to defined areas. However, in five patients with variousdegrees of intestinal metaplasia, present in an islet-like fashion inthe gastric mucosa, those lesions were strongly stained by MAb 17-1A andthe four new MAbs. The 17-1A Ag was also distinctly expressed in 9 of 9gastric carcinomas tested.

DISCUSSION

[0053] We have demonstrated that the 17-1A Ag is a glycoproteinmigrating in SDS-PAGE with an apparent molecular weight of 37 kD undernon-reducing conditions. Comparative analysis of three different coloncarcinoma lines did not indicate a hetereogeneity of the 17-1A Ag whennon-reducing conditions were applied. After reduction of the 17-1Aprecipitates with 2-mercaptoethanol and separation by SDS-PAGE a 33 kDband appeared which could be found with all the cell lines tested. Inaddition, a 40 kD band could be seen as a major component in the DLD-1cell lysate and to a lesser degree in the lysate of HT-29 cells. This 40kD band was virtually absent in lysates from WiDr cells. Whenprecipitates from DLD-1 or HT-29 cells were processed in singleexperiments and analyzed in a parallel fashion under reducing andnon-reducing conditions, again two bands of 33 and 40 kD appeared in thepresence, but only a single band of 37 kD in the absence of2-mercaptoethanol. Thus, reduction of the 17-1A Ag may result in theformation of two new forms of the molecule with differentelectrophoretic mobilities. Most probably the presence of intrachaindisulfide bonds may account for this peculiar migration behavior.Alternatively, the 17-1A Ag may actually exist as a dimer of proteinsexhibiting the same migration behavior under non-reducing conditions.The absence of the 40 kD molecule in WiDr lysates may then be explainedby a differential accessability of this protein forlactoperoxidase-mediated iodination in different cell lines. Furtheranalysis using metabolic labelling and cross-linking chemicals may berequired to solve this question. Ross et al. have recently described acarcinoma-associated surface glycoprotein defined by their MAb GA733,that consists of 30 kD and 40 kD subunits. They suggest that the 17-1Aantibody recognizes the same antigen, but binds to a different epitopethan MAb GA733. Direct comparisons with the antibodies described hereshould reveal the relationship of these antigens.

[0054] The four new anti-17-1A Ag MAbs which we obtained displayed atissue reactivity comparable to the original 17-1A antibody in normalorgans and in tumors. So far no differential epitope expression wasfound in a variety of tissues examined. Two of these MAbs (M72 and M74)recognize a determinant closely related to that detecterd by MAb 17-1Aas judged by cross-blocking experiments, whereas the MAbs M77 and M79apparently define at least one additional epitope since they did notinhibit binding of the biotinylated 17-1A antibody. Interestingly, theepitope spcecificity of these MAbs was correlated with their reactivitywith a goat anti-idiotypic antiserum. These MAbs will be of interest foranalyzing their biological activities in conjunction with the original17-1A antibody.

[0055] The 17-1A Ag is broadly expressed in non-malignant epithelialorgans and is also present in most carcinomas originating from17-1A-positive tissues. Quantitative differences in expression betweennormal and malignant tissue were not evident in a comparativeimmunohistochemical analysis of colorectal carcinomas and normal mucosaobtained from the same patietns. However, a possible structuralheterogeneity of this epithelial antigen on normal and malignant cellsis not ruled out by the present analysis.

1. A method of treating gastrointestinal tumor comprising administeringto a patient afflicted with a gastrointestinal tumor, a murinemonoclonal antibody which specifically binds to an epitope of 17-1Aantigen, the antibody being administered parenterally in multiple dosesof 100 milligrams or more per dose for a total dose of about 0.1-5 gramsof antibody.
 2. A method of claim 1, wherein the gastrointestinal tumoris gastrointestinal adenocarcinoma, colorectal or pancreatic carcinoma.3. A method of claim 1, wherein a mixture of two or more murinemonoclonal antibodies which specifically bind an epitope of the 17-1Aantigen are administered.
 4. A method of claim 1, wherein a dose ofantibody is infused intraveneously.
 5. A method of claim 1, wherein thetotal dose is about 1-5 grams.